Cold insoluble globulin (CIG) is a plasma protein that coprecipitates with fibrinogen and other proteins found in the cryoprecipitate of plasma and related fractions. It appears that CIG has been reported in the literature under a variety of names including, for instance, .alpha..sub.2 -glycoprotein, non-immune .alpha..sub.2 -opsonic macroglobulin, fibronectin or plasma fibronectin, human plasma cryoprecipitate, humoral recognition factor, antigelatin factor, CIg, LETS protein, .alpha..sub.2 -surface binding macroglobulin.
It has been suggested that CIG possesses opsonic activity. Opsonins are serum factors that promote the phagocytosis of foreign particulate matter.
Phagocytosis is the specialized function of certain cells in higher organisms involved in host defense with the purpose of ingesting and digesting harmful particles such as bacteria, antigen-antibody complexes, tissue debris and colloidal pollutants. This process depends on humoral recognition factors (opsonizing factors) which give it selectively and efficiency (DiLuzio et al., Advan. Exp. Med. Biol. 15, 373-390 (1971)).
The reticuloendothelial system (RES) is involved in the phagocytic clearance and subsequent intracellular degradation of both blood-borne foreign and endogenous effecte, particulate, or denatured matter. This vascular clearance capacity of the RES is primarily mediated by sessile phagocytic cells which line the vascular compartment in the linear, spleen, lung, and bone marrow. The reticuloendothelial system is a defense mechanism which when operating efficiently allows higher animal forms to survive severe stress such as trauma, sepsis, surgery, etc. Reticuloendothelial cells are found, for example, fixed in the liver, spleen, bone marrow and lymph nodes as well as circulating in the vascular tree and tissue spaces. The major portion of the RES consists of sessile macrophages. Due to their strategic anatomic locations and intense phagocytic activity they filter the blood system to protect the pulmonary and systemic vascular beds from microembolism and trauma caused by accumulation of such products. As a clearance mechanism, they remove from the blood bacteria, denatured proteins, immune complexes, microaggregates of fibrin, injured platelets, viruses, tumor cells, endotoxins, lysosomal enzymes, other toxic and/or antigen bloodborne particulate matter, and the like.
Through their opsonic activity, a variety of proteins have been shown to possess the ability to promote the phagocytic activity of the reticuloendothelial cells. These proteins include several .alpha..sub.2 -opsonic glycoproteins, antibodies, opsonically active fragments of the complement system and C-reactive protein. There are distinct differences among these proteins with regard to chemical characterization and substrate specificity. One of the .alpha..sub.2 -opsonic glycoproteins, plasma fibronectin, has been considered to be a non-specific thermolabile factor involved in mononuclear phagocytosis of foreign colloids, denatured protein, leukemic leukocytes and certain tumor cells, with minimal documented importance in bacterial phagocytosis. By contrast, antibodies or immunoglobulins, certain components of the complement system and C-reactive protein are considered to be specific and are actively involved in the opsonic mediated phagocytosis by both mononuclear and polymorphonuclear leukocytes. As stated by Molnar et al, Biochem. Biophys. Acta, 493, 37-54 (1977), the best studied opsonins are antibodies and certain complement components.
Molnar et al., supra, suggested that in the rat, non-specific opsonin could be involved as a first line of defense against invading microorganisms in a non-specific manner. This mechanism has been proposed to be essential for survival until the immune system takes over. According to Blumenstock et al., J. Reticuloendothel Soc., 19, 157-172 (1978), non-specific stimulation of the RES results in a state of hyperphagocytosis that can be quantified by blood clearance of test colloids. This hyperphagocytosis has been correlated with increased resistance to infection, malignant disease and traumatic shock. Experimental induction of RES phagocytic depression caused by colloid induced blockage decreases the resistance to such insults. Following burn shock or traumatic injury, the consumptive depletion of both specific and non-specific opsonic proteins appears to predispose higher animals to septicemia and/or microvascular collapse, i.e., shock.
Studies have indicated that the function of the reticuloendothelial system is related to survival after severe trauma and shock. Thus, a reduced RE function has resulted in a markedly depressed tolerance to trauma in animals while an enhanced tolerance to trauma has been found in animals with RES hyperactivity.
Van Oss et al., Immunol. Commun., 3, 329-335 (1974) found that .alpha..sub.2 -glycoprotein (.alpha..sub.2 HS glycoprotein) was decreased to 25 to 50% of normal values in each of eight trauma patients studied. This glycoprotein appeared to act directly on the surface of microorganisms and they considered it a true aspecific opsonin. They felt that the decreased concentration of this protein in trauma patients was a likely factor contributing to the decreased resistance to bacterial infections. They also noted another factor, an .alpha..sub.1 acid glycoprotein, which may be an inhibitor of phagocytosis.
Scoville et al., J. Trauma, 16, 898-904 (1976) reported on 20 consecutive patients with multiple injuries from motor car accidents or blunt trauma, each of whom had a documented episode of hypotension. Plasma opsonic activity (detected using a liver in vitro bioassay) was decreased markedly following trauma. The initial post-traumatic hypoopsonemia was more severe in the non-surviving patients than in the surviving patients. Survivors, following trauma, manifested restoration of opsonin levels with a definite transient rebound hyperopsonemia during the recovery phase. Non-surviving patients exhibited persistant systemic .alpha..sub.2 -globulin opsonic deficiency. They concluded their report with the following statement, "the importance of posttrauma RES dysfunction to survival following severe injury warrants further investigation and clinical consideration".
Surgery has induced alterations in plasma fibronectin concentrations. DiLuzio and Lindsey, Proc. Soc. Exp. Biol. Med., 143, 715-718 (1973) evaluated the influence of surgical stress plasma opsonin levels in both renal transplant donors and recipients by the in vitro liver slice bioassay. Both surgical populations showed significant depletion of opsonin activity at 1-7 days post-surgery. Restoration occurred in all cases.
Saba and Scovill, Surg. Annu., 7, 71-102 (1975) reported that following surgical intervention there was a transient but profound depression in RE phagocytosis mediated by a deficiency in circulating plasma opsonic capacity. Eventual restoration of RE function during the later post-operative period could be correlated with a recovery of the blood opsonic levels. Following major accidental or surgical trauma a failure in systemic RE phagocytosis was observed. These authors suggest that this occurrence may be significant to host defense against septicemia. For a transient period of the post injury interval, this failure may increase the susceptibility of the host to pulmonary microvascular localization of denatured protein such as fibrin as well as to embolization of fat globules and blood-borne tumor cells. They concluded that with the isolation of the humoral factor (opsonic protein), this tool would have both diagnostic as well as prognostic value as an humoral index of the functional state of the RES. Such values would be helpful prior to and following surgery or during the course of clinical therapy for malignancy and traumatic shock.
Aronsen et al., Scand. J. Clin. Lab. Invest. 29, suppl. 124: 127-136 (1972) measured cold insoluble globulin (CIG) in plasma following cholecystectomy. CIG showed an initial rapid but small drop in concentration followed by a slow increase with a concentration maximum by one week post-operation and then a fall to normal. An electroimmuno precipitation assay was used to determine the concentrations with the mean determined as a percentage of that found for normals. The pre-operative mean value was 96% with a standard deviation (S.D.) of 22 and a range of 58 to 135. However, the mean at post-operative day 2 was 79% with a S.D. of 23 and a range of 35 to 120.
Kaplan and Saba, J. Reticuloendothel Soc., 15, 682 (1974) found a 76% decrease in opsonin levels and an associated RE depression 60 minutes post-surgery (celiotomy) in rats. Purified .sup.125 I labeled opsonin protein was found to localize at the incision site.
Kaplan et al., (1976) suggested that the accumulation of opsonic protein at the site of surgery was the result of affinity for damaged tissue. They also noticed a shorter biological halflife for opsonic protein in operated animals than found in normal rats (134 minutes in normals). They concluded that the relationship between the level of circulating opsonic protein and the post-operative RES defense in terms of non-specific clearance of microaggregates and foreign abnormal cells from the blood warrants investigation.
Molnar et al., supra (1977) labeled membrane fragments from homogenized tissues and showed that these fragments were taken up by liver slices. This uptake was dependent on the concentration of the rat .alpha.-2-opsonin.
CIG was first partially purified in a biologically active form by Mosesson and Umfleet, J. Biol. Chem., 245, 5728-5736 (1970). They suggested in this report that the CIG (plasma fibronectin) level in serum is somewhat lower than that in plasma due to incorporation in the fibrin clot. An improved method for purifying CIG is reported by Chen et al., Analyt. Chem., 79 144-151 (1977).
Mosher, J. Biol. Chem., 250, 6614-6621 (1975), based on in vitro data, suggests that in the presence of activated plasma factor XIIIa (fibrin stabilizing factor) CIG can be covalently cross-linked to fibrin alpha chain or to other CIG molecules. Mosher, J. Biol. Chem., 251, 1639-1645 (1976) suggested that a clot formed from normal plasma would contain 94% fibrin and 6% CIG assuming 50% of the CIG is cross-linked to fibrin. Mosher, Thromb. Res., 9, 37-45 (1976) further reported decreased levels of CIG in monkeys that developed disseminated intravascular coagulation following infection with Rocky Mountain spotted fever. Mosesson et al., supra (1975) reported that some of his unpublished work suggests that CIG levels tended to be reduced in a significant proportion of patients manifesting the disseminated intravascular coagulation syndrom.
Recently, Blumenstock et al, J. Biol. Chem., 253, 4287-4291 (1978) suggested that human CIG (or plasma fibronectin) showed immunological identity with their human opsonin preparations. Although recognizing the fact that proof of biological identity would require a demonstration that highly purified CIG is opsonically active, no evidence could be obtained because isolation of CIG by ethanol fractionation (as reported by Mosesson et al., Biochem. Biophy. Acta, 386, 509-524 (1975)) yielded an inactive product.
Thus, it can be seen that a biologically active, highly purified CIG is desireable to characterize its opsonic activity and to aid in quantifying opsonic levels in the blood for diagnostic and therapeutic purposes.